A. Specific Aims: The overall goal of this proposal is to use reversible immortalization approach to establish cell lines derived from primary human renal proximal tubule epithelial cells (RPTECs) and hepatocytes. Liver and kidney are two critical organs for drug metabolism and excretion, and are frequently affected by drug-induced toxicity. Currently, no suitable human kidney cell line is available for drug screening in the early stages of drug discovery and development. While primary human hepatocytes are available, the high cost to culture such cells precludes their use in the drug screening process. If immortalized cell lines that are phenotypically stable and retain all or most of the primary cell functions can be established, they can be used for high-throughput screening of potential drugs. There are three specific aims in this proposal. I. Characterization of TH1 and TH7 cells for kidney functions: In our preliminary study, we used human immunodeficiency virus (HIV)-based vectors containing the cDNAs encoding the SV40 T antigen (Tag) and the catalytic subunit of human telomerase (hTERT) to establish several immortalized cell lines derived from primary human RPTECs. We propose to characterize the biochemical and functional activities in two of these cell lines, TH1 and TH7. We will determine the basal expression and the activity of xenobiotic metabolizing enzymes in these two cell lines. We will evaluate the activity of drug transport proteins, drug uptake and efflux. We also propose to use Cre-LoxP mediated recombination system to remove the introduced Tag and the hTERT cDNAs from these two cell lines and examine its effect on cell proliferation, differentiation and biochemical functions. II. Establishment and characterization of immortalized human hepatocytes: We propose to use the same strategy as described in specific aim I to establish and characterize immortalized human hepatocytes. Ill. Evaluation of drug toxicity in the immortalized cell lines: We will determine the drug sensitivity of the established cell lines, including methotrexate (MTX), cisplatin, cyclophosphamide and paclitaxel. The toxicity induced by these drugs in primary human cells and in cell lines derived from hepatocytes and RPTECs will also be examined and the results will be compared with those from the immortalized cell lines established in this current study.